Occurrence of Neospora caninum in dogs and its correlation with visceral leishmaniasis in the urban area of Campo Grande, Mato Grosso do Sul, Brazil

Neospora caninum is an obligate intracellular protozoan that can infect domestic and wild canids, as well as ruminants and equines, and is described as causing neuromuscular alteration and death in dogs. Visceral leishmaniasis (VL) is an infectious disease that affects both humans and animals, being caused by protozoan parasites of the genus Leishmania, of which Leishmania (Leishmania) chagasi is found in Brazil -- transmitted by sand flies, such as Lutzomyia longipalpis, in most of the American continent. The immunosuppression caused by VL can promote the occurrence of co-infections with other agents. In order to determine the frequency of N. caninum and its relationship to VL in Campo Grande, MS, Brazil, 345 blood sera were collected from dogs. The sera were submitted to an indirect fluorescent antibody test (IFAT) for detection of anti-N. caninum antibodies and VL antibodies. N. caninum was found in 26.5% of VL-negative dogs and in 29% of VL-positive ones. Among males, it was found in 30.7% of VL-negative animals and in 30.4% of VL-positive ones; among females, in 21% of VL-negative animals and in 27.7% of VL-positive ones. Among juvenile dogs (under 1 year), N. caninum was detected in 10.5% of VL-negative animals and in 11.2% of VL-positive ones. For adult dogs (1 year and older) the results were 31.4% for VL-negative animals and 28.8% of VL-positive ones. The study revealed a statistically significant association with age (chi(2)=9.76, P<0.05) in the N. caninum results for VL-negative animals. No significant correlation in N. caninum seroprevalence was found when VL-positive or VL-negative dogs were compared (chi(2)=0.21, P=0.64). The findings suggest that in Campo Grande N. caninum and VL co-infection is common in dogs, though VL does not appear to enhance susceptibility to N. caninum.     

Identification of candidate markers on bovine chromosome 14 (BTA14) under milk production trait quantitative trait loci in Holstein

The objective of this study was to identify single-nucleotide polymorphisms using a bovine chromosome 14 high-density SNP panel after accounting for the effect of DGAT1. Linkage disequilibrium information and sire heterozygosity were used to select markers for linkage analysis on bovine chromosome 14 for milk production traits in 321 Holstein animals. Results show putative milk peaks at 42 and 61 cM, both at p<0.10, a fat yield peak at 42 and 63 cM, both at p<0.05; a protein yield peak at 42 (p<0.01) and 84 cM (p<0.05); fat per cent peaks at 3 (p<0.01) and 29 cM (p<0.05), and a protein per cent peak at 4 cM (p<0.05). Once quantitative trait loci positions were established, allele substitution effects for all markers were evaluated using the same statistical model. Overlaying information between quantitative trait loci (QTL) and allele effect analysis enabled the identification (p<0.01) of 20 SNPs under the milk yield QTL, 2 under both of the fat yield peaks, 8 and 9 under the protein yield peaks, 2 and 6 for the fat per cent peaks and 5 for the protein per cent peak. One SNP in particular, ss61514555:A>C, showed association with 3 of the 5 traits: milk (p=1.59E-04), fat (p=6.88E-05) and protein yields (p=5.76E-05). Overall, combining information from linkage disequilibrium, sire heterozygosity and genetic knowledge of traits enabled the characterization of additional markers with significant associations with milk production traits.     

Transmission of Leishmania infantum via blood transfusion in dogs: potential for infection and importance of clinical factors

Blood transfusion is an important routine practice in veterinary medicine that generally involves the use of whole blood. Permanent blood donors must be vaccinated against viral infections that affect dogs and submitted periodically to clinical and serological examinations to detect blood-transmitted diseases. There is a very high risk of transmission of infectious agents, particularly protozoans due to their long incubation periods, subclinical persistence in infected animals and likelihood of remaining viable in bloodstocks. The aim of the present study was to identify the potential of asymptomatic and oligosymptomatic dogs for Leishmania infantum transmission as a result of transfusional practice. Nineteen Leishmania-seropositive adult dogs of both sexes and indeterminate breeds were selected as donors. The animals were classified as symptomatic, oligosymptomatic or asymptomatic after clinical examination and evaluated by ELISA, IFAT and bone marrow puncture biopsies. Whole blood and monocyte cells were collected and used for dog's serological evaluation and inoculation in culture medium as well as in hamsters. All but three dogs were positive for IFAT, ELISA and parasite demonstration in bone marrow aspirates, irrespective of their clinical conditions. Parasites were detected in 77% of the whole blood and 90% of the monocyte cultures. Six months after inoculation with whole blood or monocytes, hamsters developed infection and clinical symptoms of visceral leishmaniasis, as well as positive titres measured by ELISA. These results suggest that blood donors should be monitored periodically and rigorously for Leishmania infection, to prevent dissemination of the disease through blood transfusion.     

Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.     

The Influence of Oxygen Tension on Cumulus Cell Viability of Canine COCs Matured in High-Glucose Medium

High in vitro oxygen (O₂) tensions are associated with enhanced levels of reactive oxygen species and cumulus oocyte complex (COC) apoptosis. The objective of this study was to determine the influence of O₂ tension on cumulus cell (CC) viability from canine oocytes. Cumulus oocyte complexes were distributed into three groups (CG, T20 and T5) and two O₂ tension levels (20% and 5%). The control group (CG) was matured in vitro in a humidified atmosphere with 5% CO₂ in air in TCM199 with 26.19 m m sodium bicarbonate, 10% (v/v) foetal calf serum (FCS), 0.10 m m gentamicin, 0.20 m m pyruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml follicle-stimulating hormone, 0.03 IU/ml human chorionic gonadotropin, and 1.0 μg/ml human somatotropin. Groups T20 and T5 were matured under 20% or 5% O₂ tensions respectively in a high-glucose medium, without FCS. T20 and T5 were as CG, and supplemented with 0.1% Polyvinyl Alcohol, and 5.5 m m glucose. After 48 h of IVM, CCs from COCs were stained with propidium iodide (1.50 m m ). The results showed that viability of CCs (cytoplasmic features and nuclear morphological integrity) was different for the three groups. Rates of apoptosis were at 57.9% (521/900) for CG, 54.4% (490/900) for T20 and 38.9% (350/900) for T5 (p < 0.001). Predominant features in apoptotic cells (n = 1361) were DNA nuclear fragments (94.0%). It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O₂ tension was efficient in reducing apoptosis in canine CCs.     

Sperm subpopulations influence the pregnancy rates in cattle

This study aimed to study the characteristics and subpopulations of spermatozoa from bulls with low and high reproductive performance based on pregnancy rates. Based on historical records of pregnancy rate from four farms, 24 bulls were selected. Two groups were established, with low pregnancy rates (n = 12; LOW), including bulls that presented pregnancy rates <52.27% (33.33% to 51.81%); and a group with high pregnancy rates (n = 12; HIGH), with pregnancy rates >52.27% (52.27% to 69.64%), after fixed-time artificial insemination (FTAI). The thawed sperm straws were analysed to sperm kinetics, morphology, plasma membrane integrity and sperm subpopulations. The LOW group exhibited a higher proportion of static cells (p < .05). In contrast, the HIGH group showed greater percentages for membrane integrity and total and progressive motility, and cells with fast and medium velocity (p < .05). In the cluster procedures, four sperm subpopulations were established. The low-fertility bulls presented the highest percentage of subpopulation 2 (41.46%), characterized by slow and progressive spermatozoa. The high-fertility bulls exhibited the highest percentage of subpopulation 3 (37.17%), characterized by fast and nonlinear spermatozoa. Results from this study indicated that bulls with greater percentages of fast and nonlinear spermatozoa seem to have greater fertilization capacity and the subpopulations analysis can be considered a tool to identify ejaculates with high fertility.     

Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.     

Fatty acid composition of intramuscular fat from Nellore steers fed dry or high moisture corn and calcium salts of fatty acids

The purpose of this work was to evaluate the fatty acid composition of the Longissimus muscle from carcasses of Nellore steers fed diets with calcium salts of fatty acids (CSFA) and high moisture corn. Forty eight steers were fed during 70 days four diets containing dry corn (DC), high moisture corn (HM), dry corn plus CSFA (DC-CSFA) or high moisture corn plus CSFA (HM-CSFA). Fatty acid composition of the Longissimus muscle was determined by gas chromatography. Corn type had no effect on the ether extract percentage and in the content of the majority of the fatty acids, although steers fed HMC showed higher levels of polyunsaturated fatty acids and polyunsaturated/saturated ratio. Feeding CSFA increased ether extract percentage but had no effect on total of saturated, unsaturated and saturated: unsaturated ratio. Both high moisture corn and calcium salts of fatty acids increased CLA (cis9, trans11) and total CLA concentrations in intramuscular fat.     

Factors Affecting Plasma Pregnancy-associated Glycoprotein 1 Concentrations Throughout Gestation in High-producing Dairy Cows

This study was designed to establish the factors, if any, which could affect plasma pregnancy-associated glycoprotein-1 (PAG-1) expression in a study population of 87 pregnant, high-producing dairy cows. The factors examined were: semen providing breed (Holstein-Friesian vs Limousin), outcome of gestation (male vs female newborn, and singleton vs twin pregnancies), lactation number, milk production at pregnancy diagnosis, plasma progesterone concentration, season of gestation (warm period, March–November vs cool period, December–February), and day of gestation (40, 90, 120, 150, 180 and 210). Pregnancy was diagnosed by transrectal ultrasound on day 40 post-insemination and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The relative contributions of the different factors on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. No significant effects of the herd, foetal sex, milk production, lactation number and plasma progesterone concentrations were observed. In contrast, twin pregnancy, the use of Limousin semen and conception during the cool period were correlated with significantly increased plasma PAG-1 concentrations throughout gestation. Our data indicate that both cow well-being during early placental development, determined in our conditions by reduced heat stress when conception occurred in the cool season, and crossbreed pregnancies lead to improved PAG-1 production throughout the gestation period.